Sulfonamide-derivative compounds for tagging nucleic acid probes

ABSTRACT

Nucleic acid hybridization probes are provided which comprise an N 4  -(substituted amino)cytosine moiety, wherein the substituted amino group comprises a tag moiety, whereby the probe is detected. Methods of preparing probes of the invention, intermediates used in such methods, and methods of using the probes of the invention in hybridization assays are also provided. Typical tag moieties employed with the probes of the invention are biotinyl, aminothiadiazole and fluorescein derivatives, connected to N 4  -amino groups of modified cytosines of the probe through linker moieties. Probes tagged with biotin are typically detected by binding to the biotinyl moieties, through a streptavidin or avidin molecule, a reporter group which includes streptavidin or avidin and then detecting a signal due to the reporter group. Probes tagged with aminothiadiazole derivatives are typically detected by essentially the same method as those tagged with biotinyl but employing as reporter group one which binds to the derivative through a carbonic anhydrase molecule. Probes tagged with fluorescein derivatives are detected by a fluorescence spectroscopic method without binding of a reporter group to the tag.

This application is a division of application Ser. No. 07/323,433, filedMar. 14, 1989, now U.S. Pat. No. 5,130,446 which is a division ofapplication Ser. No. 07/122,496, filed Nov. 16, 1987, now U.S. Pat. No.4,833,251, issued May 23, 1989, which is a continuation of applicationSer. No. 06/748,499, filed Jun. 25, 1985, now abandoned.

TECHNICAL FIELD

The present invention relates to nucleic acid hybridization probes and,more particularly, to the chemical labeling of nucleic acids to makethem useful as hybridization probes.

BACKGROUND OF THE INVENTION

The use of single-stranded DNA or RNA probes, to test for the presenceof particular DNAs or RNAs and associated biological entities in samplesof biological material, is well known. See, e.g., Grunstein and Hogness,Proc. Nat'l. Acad. Sci. (US) 72, 3961-3965 (1975); Southern, J. Mol.Biol. 98, 503-505 (1975); Langer et al., Proc. Nat'l. Acad. Sci. (US)78, 6633-6637 (1981); Falkow and Moseley, U.S. Pat. No. 4,358,535; Ward,et al., European Patent Application Publication No. 0 063 879;Englehardt, et al., European Patent Application Publication No. 0 097373; Meinkoth and Wahl, Anal. Biochem. 138, 267-284(1984).

Among areas in which such probes find application are testing of foodand blood for contamination by pathogenic bacteria and viruses;diagnosis of fungal, bacterial and viral diseases by analysis of feces,blood or other body fluids; diagnosis of genetic disorders, and certaindiseases such as cancers associated with a genetic abnormality in apopulation of cells, by analysis of cells for the absence of a gene orthe presence of a defective gene; and karyotyping. See Klausner andWilson, Biotechnology 1, 471-478 (1983); Englehardt, et al. supra; Wardet al., supra; Falkow and Moseley, supra.

The principle which underlies the use of such probes is that aparticular probe, under sufficiently stringent conditions, will, viahydrogen-bonding between complementary base moieties, selectivelyhybridize to (single-stranded) DNA or RNA which includes a sequence ofnucleotides ("target sequence") that is complementary to a nucleotidesequence of the probe ("probing sequence" specific for the targetsequence). Thus, if a biological entity (e.g., virus, microorganism,normal chromosome, mammalian chromosome bearing a defective gene) to betested for has at least one DNA or RNA sequence uniquely associated withit in samples to be tested, the entity can be tested for using a nucleicacid probe.

A DNA or RNA associated with an entity to be tested for and including atarget sequence to which a nucleic acid probe hybridizes selectively ina hybridization assay is called "target" DNA or RNA, respectively, ofthe probe.

A probe typically will have at least 8, and usually at least 12,ribonucleotides or 2'-deoxyribonucleotides in the probing sequence thatis complementary to a target sequence in its target DNA or RNA. Outsidethe probing sequences through which a probe complexes with its targetnucleic acid, the probe may have virtually any number and type of bases,as long as the sequences including these additional bases do not causesignificant hybridization with nucleic acid other than target nucleicacid under hybridization assay conditions. That is, a probe will bespecific for its target DNA or RNA in hybridization assays.

To be useful in analyzing biological samples for the presence of atarget DNA or RNA, a polynucleotide probe must include a feature whichwill render detectable the duplex formed when the probe is hybridized toits complementary sequence in the target (single-stranded) DNA or RNA.Typically, such features in a probe include radioactive atoms orpyrimidine or purine bases chemically modified to include moieties whichare readily and sensitively detected by any of a number of techniques.

For example, a probe may be made with ³² P-labelled nucleosidetriphosphates; then the probe itself, as well as target DNA or RNA withthe probe hybridized to it, can be detected by means of radiation from³² P-decay.

Probes whose detectability is based radioactive decay are unsuitable formany applications because of safety problems and licensing requirementsassociated with radioactive materials and because of degradation of theprobes that occurs with radioactive decay during storage. Thus, probeswhose detectability is based on chemical modification of pyrimidine orpurine bases are preferred in many situations.

There are numerous examples of modified purine or pyrimidine bases inprobes wherein a moiety, herein referred to as a "tag moiety," ischemically linked to render detectable target DNA or RNA hybridized withprobe. See, e.g. Ward et al., supra; Englehardt et al., supra; Klausnerand Wilson, supra. Typically, the "tag moiety" is a moiety to which aprotein will bind with high affinity, e.g. an antigen to which anantibody binds; a biotinyl or iminobiotinyl moiety to which avidin orstreptavidin will bind; an inhibitor of an enzyme to which the enzymebinds. A protein which binds with high affinity to a tag moiety of aprobe is referred to herein as a "conjugate protein" of the tag moiety.

In a typical assay, after probe is hybridized to target DNA or RNA, a"reporter group" is added to the system and binds to the tag moiety ormoieties of the hybridized probe. A "reporter group" provides a signalwhich renders detectable the probe that is hybridized to target DNA orRNA. A typical reporter group is a conjugate protein of the tag moietyor a complex, involving such a conjugate protein, which binds to tagthrough the binding site for tag in the conjugate protein. The reportergroup so bound is then detected by an appropriate immunological,physical, or biochemical technique. For example, if the reporter groupis simply a conjugate protein, detection might be by any of a number ofwell known immunoassay techniques, based on antibodies directed againstthe conjugate protein. If the reporter group is a conjugate proteinwhich naturally contains a chromophore or fluorophore, or is a conjugateprotein modified to include such a moiety, detection might be by aspectroscopic technique based on the chromophore or fluorophore. If thereporter group is a heteropolymer or homopolymer of enzymes, including aconjugate protein, detection could involve detection of substancesproduced by enzymatic reactions catalyzed by enzymes in the polymer.Ward et al., supra, Englehardt, et al., supra, and Klausner and Wilson,supra, describe a number of techniques for assaying reporter groupsbound to tag moieties of probes.

A tag moiety itself, without being bound by a reporter group, mightprovide detectability to a probe. For example, a tag moiety which is afluorophore or chromophore can be detected with a suitable spectroscopictechnique without binding of a reporter group. See, e.g., Bauman et al.,J. Histochem. Cytochem. 29, 227-237(1981).

In some cases wherein pyrimidine or purine bases are chemically modifiedby the addition of a tag moiety, a linking moiety will separate the tagmoiety from the site of modification on the pyrimidine or purine base.See, e.g., the Ward et al. and Englehardt et al. references, supra. Insome cases, such linking moieties facilitate the attachment of tagmoieties to probe. Further, a linking moiety tends to hold a tag moietysome distance from the modified purine or pyrimidine base, therebyincreasing accessibility of the tag moiety to binding by a reportergroup and, further, reducing interference with formation or stability ofduplexes between probe and target DNA or RNA in those instances wherethe tag moiety has a large molecular weight.

Polynucleotide probes which comprise at least one cytosine moietymodified to have a tag moiety linked, directly or through a linkingmoiety, to the N⁴ -position, have not been available heretofore.

Because the amino group at the 4-position of cytosine is involved inhydrogen-bonding between cytosine and guanine moieties in nucleic acidduplexes, it has been thought heretofore that modifications to thisamino group would be unacceptable in nucleic acid probes. It has beenthought that such modifications in a nucleic acid would interfere withduplex formation, and thereby result in a probe with unacceptablespecificity and sensitivity, by severely disrupting guanosine-cytosinehydrogen-bonding. See Ward et al., supra; Ruth, Patent CooperationTreaty International Publication No. W084/03285(1984).

The chemistry of modifying cytosine moieties at the N⁴ -nitrogen hasbeen studied with cytidine and 2'-deoxycytidine and their phosphates,both as monomers and included in single-stranded polynucleotides. Nittaet al., FEBS Letters 166, 194-198 (1984); Negishi et al. (I), Nucl.Acids Res. Symp. Series I2, pp. 29-30(1983); Negishi et al., (II), Nucl.Acids Res. 11, 5223-5333 (1983); Hayatsu, Prog. Nucleic Acid Res. andMol. Biol. 16, 75-124 (1976).

The N⁴ -amino group, in N⁴ -aminocytidine and N⁴ -amino-2'-deoxycytidineand their phosphates, both as monomers and included in single-strandedpolynucleotides, is known to have reactivities characteristic ofsubstituted hydrazines and reacts accordingly with aldehydes, ketones,isothiocyanates and imidates. Nitta et al., supra; Negishi (I), supra;Hayatsu, supra. See also P. Smith, The Chemistry of Open-Chain OrganicNitrogen Compounds, W. A. Benjamin Inc., New York, N.Y., Vol. II, pp.119-209 (1966).

Nitta et al., supra, have reported transamination of cytosine moietiesin polycytidine with hydrazine in the presence of bisulfite; andderivatization of the transaminated product with an adduct ofglutathione with pyruvic acid, wherein the adduct reacts through theketo-carbon of pyruvate with the N⁴ -amino group.

SUMMARY OF THE INVENTION

We have discovered nucleic acid probes which comprise a cytosine moietymodified to N⁴ -(substituted amino)cytosine, wherein the substituent onthe amino group comprises a tag moiety.

We have discovered, further, polynucleotides, with cytosines modified toN⁴ -amino cytosines, which are intermediates in the syntheses of thepolynucleotide probes of the present invention. We have also discoverednovel compounds, which can be used to link certain tag moieties topolynucleotides comprising N⁴ -aminocytosine moieties to makepolynucleotide probes according to the invention, and novel methods tomake probes of the invention which comprise reacting the novel compoundswith polynucleotides comprising N⁴ -aminocytosine moieties.

Duplexes between the probes of the invention and target DNA and RNA towhich they bind are also part of our discovery, as are the duplexescomplexed with reporter groups.

In yet another aspect of the invention, we have discovered a novelmethod for testing a sample for the presence of a biological entity,associated with a target DNA or RNA. Such method comprises combiningsingle-stranded DNA or RNA, derived from the sample, with a nucleic acidprobe of the invention specific for the target DNA or RNA. Reactionconditions for carrying out the method are selected whereby stableduplexes form between probe and at least a portion of its target nucleicacid but significant duplex formation between probe and non-targetnucleic acids present in the sample is excluded. The method alsocomprises detecting said stable duplexes by means of a signal from thetag moiety directly or from a reporter group, which is bound to the tagmoiety.

Finally, our invention encompasses kits for testing samples for thepresence of a biological entity associated with target DNA or RNA, whichwill be present in a sample only if the biological entity is present.Such kits comprise a polynucleotide which has a sequence complementaryto a sequence within the target DNA or RNA but not other DNA or RNA insamples to be tested. In one embodiment, the kits include, in additionto the polynucleotide, reagent to convert cytosine moieties in thepolynucleotide to N⁴ -aminocytosines. In a further embodiment, the kitscomprise a probe according to the invention, with cytosines of thepolynucleotide modified the N⁴ -(substituted amino)cytosines wherein thesubstituent on the amino group includes a tag moiety.

DETAILED DESCRIPTION OF THE INVENTION

In one aspect, the present invention is a nucleic acid probe whichcomprises a modified cytosine moiety of Formula I ##STR1## wherein R₁ is(i) --N═C(R₂)--R₅ --R₆,

(ii) --NH--(CHR₂)--R₅ --R₆, or

(iii) --NH(C═R₃)NH--R₅ --R₆,

wherein R₂ is hydrogen or alkyl of 1 to 4 carbon atoms; R₃ is sulfur oroxygen; R₅ is a linker moiety; and R₆ is a tag moiety.

The nitrogen atom, at the terminus of the R₁ groups of Formula I that isbonded to the N⁴ -position of cytosine, is the nitrogen atom of thegroup referred to herein as the N⁴ -amino group of N⁴ -aminocytosinemoieties.

In another aspect, the invention includes duplexes formed between such aprobe and its target DNA or RNA in a sample tested with the probe. Theinvention includes, further, such duplexes complexed with a reportergroup conjugate to the tag moiety on the probe.

In yet another aspect, the invention relates to a method for testing asample for a target DNA or RNA which comprises (i) combining, with thetarget DNA or RNA, a nucleic acid probe specific for the target DNA orRNA, said probe comprising a modified cytosine moiety of Formula I##STR2## wherein R₁ is (i) --N═C(R₂)--R₅ --R₆,

(ii) --NH--(CHR₂)--R₅ --R₆, or

(iii) --NH(C═R₃)NH--R₅ --R₆,

wherein R₂ is hydrogen or alkyl of 1 to 4 carbon atoms; R₃ is sulfur oroxygen; R₅ is a linker moiety; and R₆ is a tag moiety; provided that thederivation of single-stranded nucleic acid from said sample and thecombining of said single-stranded nucleic acid with said probe are underconditions whereby stable duplexes form between the probe and at least aportion of the target nucleic acid but not significantly between theprobe and non-target DNA or RNA; and (ii) determining whether stableduplex was formed in step (i) by (a) separating unduplexed probe fromduplexed probe formed in step (i); (b) if the tag moiety, R₆, providesdetectability through binding thereto of a reporter group, combining theduplexed probe with a reporter group conjugate to said tag moiety, underconditions whereby the reporter group binds to at least a portion of anyof said tag moiety that is present, and then separating from the productso treated substantially all reporter group not bound to said tagmoiety; (c) treating the product of step (i), after treatment accordingto step (ii)(a) and, if used, step (ii)(b), to produce a signal from anyof said tag that is present, if tag without bound reporter groupprovides a signal to render probe detectable, or from any of said boundreporter group that is present; and (d) determining whether a detectablesignal is generated by the treatment of step (ii)(c). The treatment ofstep (ii)(c) will depend on the tag moiety or reporter group which is toprovide the signal and on the type of signal to be provided. If, forexample, the signal is to be fluorescence emission, the treatment can besimply exposure to electromagnetic radiation of wavelength suitable tostimulate the emission from the tag moiety or reporter group. To citeanother example, if the signal is to be the appearance of a visiblecolor due to a reaction catalyzed by an enzyme which is part of areporter group, the treatment will involve combination of the product ofstep (i), after treatment according to steps (ii)(a) and (ii)(b), withsubstrates for the enzyme and any other compounds necessary to producethe substance which provides the color. The determination of step(ii)(d), of whether a detectable signal is generated, will involveobservation, with the naked eye or with suitable instrumentation ifnecessary, to detect any signal that might be generated. Also, usuallythe method will be carried out in parallel on the sample being tested(test sample), a sample of nucleic acid known to be free of DNA or RNAwith target segment of the probe (negative control), and possibly also asample known to contain DNA or RNA with target segment of the probe(positive control). When the method is so carried out, in parallel ontest sample, negative control and positive control, the determinationwill then involve comparing the signals from the samples to ascertainthat the assay system was functional (producing a signal from thepositive control that is greater than "background" signal, from thenegative control) and whether the signal detected from the test samplewas greater than "background" signal. When signal from test sample isdetermined to be greater than background signal, the test sample signalcan be ascribed to target DNA or RNA in the test sample.

The invention entails also a number of kits for testing samples for thepresence of a biological entity that is associated with a target DNA orRNA which is present in the sample only if the biological entity ispresent. One of said kits comprises (i) a quantity of a nucleic acidwhich comprises a cytosine moiety and has a sequence of a probe specificfor said target DNA or RNA and (ii) reagents to convert at least aportion of the cytosine moieties in said quantity of nucleic acid to N⁴-aminocytosine moieties. Another of said kits comprises a nucleic acid,which comprises an N⁴ -aminocytosine moiety and has a sequence of aprobe specific for said target DNA or RNA. Yet another of the kitsaccording to the invention comprises a nucleic acid probe specific forsaid target DNA or RNA, which probe comprises a modified cytosine moietyof Formula I ##STR3## wherein R₁ is (i) --N═C(R₂)--R₅ --R₆,

(ii) --NH--(CHR₂)--R₅ --R₆, or

(iii) --NH(C═R₃)NH--R₅ --R₆,

wherein R₂ is hydrogen or alkyl of 1 to 4 carbon atoms; R₃ is sulfur oroxygen; R₅ is a linker moiety; and R₆ is a tag moiety.

The invention entails further a compound of ##STR4## wherein --R₅₁ -- is--NH-- or --NH--N═C(R₅₁₁)--, wherein the amino group is bonded to thecarbonyl group and wherein R₅₁₁ is hydrogen or alkyl of 1 to 4 carbonatoms; wherein R₅₂ is --CO₂ R₅₃, --(CO)R₅₄, ##STR5## wherein R₅₃ isalkyl of 1-5 carbon atoms; wherein R₅₄ is hydrogen, alkyl of 1-5 carbonatoms or chloro; wherein R₅₅, R₅₆ and R₅₇ are the same or different andare each alkyl of 1-6 carbon atoms; wherein R₅₈ and R₅₉ are the same ordifferent and are each alkyl of 1-5 carbon atoms; wherein R₆₀ isalkylene of 2 or 3 carbon atoms; wherein m is 2 to 20; wherein X₅₀ is Sor S═O; and wherein X₅₂ is O or NH.

The invention includes also a compound of Formula XXXII ##STR6## whereinR₅₈ and R₅₉ are the same or different and are each alkyl of 1-5 carbonatoms; wherein R₆₀ is alkylene of 2 or 3 carbon atoms; and wherein k is2 to 20.

"Halogen" means fluoro, chloro, bromo or iodo.

Another compound of the invention is of Formula XLV:

    R.sub.45 (NH)(C═R.sub.46)(NH)(CH.sub.2).sub.j R.sub.47 XLV

wherein R₄₅ is fluorescein, tetramethylrhodamine, ortetraethylrhodamine; R₄₆ is oxygen or sulfur R₄₇ is --CHO, --CO₂ H,--CH₂ OH, ##STR7## wherein R₅₈ and R₅₉ are the same or different and areeach alkyl of 1-5 carbon atoms; wherein R₆₀ is alkylene of 2 or 3 carbonatoms; and wherein j is 2 to 20.

Compounds of Formulas LII, XXXII, and XLV are intermediates inpreparations of preferred probes of the invention.

The present invention also entails processes for making compounds ofFormulas LII, XXXII, and XLV. These processes are taught in examplesbelow.

The invention entails further a process for making a preferred nucleicacid probe of the invention, which comprises a modified cytosine moietyof Formula LIX ##STR8## wherein X₅₀ is S or S═O; wherein X₅₂ is O or NH,wherein --R₅₁ -- is --NH-- or --NH--N═C(R₅₁₁)--, wherein the amino groupis bonded to the carbonyl group and wherein R₅₁₁ is hydrogen or alkyl of1 to 4 carbon atoms; and wherein m is 2 to 20, which process comprisesreacting a nucleic acid, with the same sequence as the probe andcomprising an N⁴ -aminocytosine moiety, with a compound of Formula LX##STR9## wherein X₅₀, X₅₂, R₅₁ and m are as in the compound of FormulaLIX. More preferred values for m are 5 to 8. When X₅₀ is S═O, X₅₂ ispreferably O. Most preferably X₅₀ is S and X₅₂ is O. Preferably R₅₁₁ ishydrogen; more preferably R₅₁ is --NH--.

The invention still further involves a process for making anotherpreferred nucleic acid probe of the invention, which comprises amodified cytosine moiety for Formula LVII ##STR10## wherein X₃₂ ishydrogen, halogen or --NO₂ ; and wherein k is 2 to 20, which processcomprises reacting a quantity of the nucleic acid with the same sequenceas the probe and comprising an N⁴ -aminocytosine moiety with a compoundof the Formula XXXIV

    R.sub.32 (CO)(CH.sub.2).sub.k CHO                          XXXIV

wherein R₃₂ and k are as in the compound of Formula LVII. More preferredvalues for k are 5 to 8. Preferably R₃₂ is theaminothiadiazole-benzolamide derivative, ##STR11## most preferably,among these derivatives, X₃₂ is hydrogen. The derivative wherein X₃₂ ishydrogen is hereinafter referred to as PABSAT (forp-amino-benzenesulfonamide with aminothiadiazole).

In another embodiment the invention involves a process for making stillanother preferred nucleic acid probe of the invention, which comprises amodified cytosine moiety of Formula XLVI ##STR12## wherein R₄₅ isfluorescein, tetramethylrhodamine or tetraethylrhodamine; R₄₆ is oxygenor sulfur; and j is 2 to 20, which process comprises reacting a quantityof the nucleic acid with the same sequence as the probe and comprisingan N⁴ -aminocytosine moiety with a compound of Formula XLVII.

    R.sub.45 (NH)(CR.sub.46)(NH)(CH.sub.2).sub.j CHO           XLVII

wherein R₄₅, R₄₆, and j are as in the compound of Formula XLVI. Morepreferred values for j are 5 to 8. Preferably R₄₅ is fluorescein ortetramethylrhodamine and R₄₆ is sulfur.

A nucleic acid with the same sequence as a probe and comprising an N⁴-aminocytosine can be provided by reacting a quantity of nucleic acidwhich has the same sequence as the probe with hydrazine in the presenceof bisulfite to convert at least a portion of the cytosine moieties insaid quantity of nucleic acid to N⁴ -aminocytosine moieties.

A nucleic acid with the sequence of a probe can be made by any ofvarious in vitro synthesis techniques known in the art followed bypurification of the nucleic acid of desired sequence by high performanceliquid chromatography (HPLC) or any other standard method known in theart. One of these techniques, involving automated solid-phase, step-wisechemical synthesis, utilizing phosphoramidate chemistry of Matteucci andCaruthers, and Beaucage and Caruthers, and purification of the nucleicacid of desired sequence by HPLC, is described in greater detail inExample I below.

When a nucleic acid with the sequence of the probe is prepared by an invitro, step-wise chemical synthesis, the sequence of the entire nucleicacid will typically be complementary to the sequence of the particularsegment of target DNA or RNA with which the probe is to hybridize in ahybridization assay. This segment of target DNA or RNA is referred to asthe target segment corresponding to the probe.

A nucleic acid with the sequence of the probe can also be prepared invivo. For example, a double-stranded nucleic acid which includes thesequence of the target segment can be isolated (as by restrictionendonuclease cleavage from the chromosomal or episomal DNA of thebiological entity to be tested for with the probe) or preparedchemically (as by step-wise solid phase synthesis of both strandsfollowed by annealing of the strands after synthesis and purification)and then cloned using standard techniques in a standard cloning vector,such as pBR322. For example, the pBR322 with the target segment insertcan be prepared in large quantities using standard preparativetechniques for plasmid DNA, which may include growth of transformed E.coli in the presence of chloramphenicol for a number of doubling timesto suppress chromosomal and enhance plasmid replication. The pBR322 withtarget segment insert can be isolated by known techniques. If targetnucleic acid is double-stranded DNA or RNA, both strands of said pBR322are useful as nucleic acid with sequence of probe. If target nucleicacid is single-stranded DNA or RNA, one or the other of the strands ofthe pBR322 will be nucleic acid with the sequence of the probe. Thechemistry described in detail below, for transamination of cytosines andattachment of tag moieties to N⁴ -amino groups of the transaminatedcytosines, can be carried out on the pBR322 in single-stranded form toprepare probe of the invention.

Another method for obtaining nucleic acid with the sequence of the probeis to insert a double-stranded segment which includes a target segmentinto RF-form DNA of a filamentous bacteriophage, such as M13mp8, M13mp9,M13mp18 or M13mp19, as are known in the art and commercially available,transforming a suitable strain of E. coli, such as E. coli JM101 orJM103, also known and commercially available, with the recombinantRF-form DNA, selecting and culturing the transformed E. coli by knowntechniques, isolating phage from the culture medium, and finallyisolating the single-stranded phage DNA from the phage by standardtechniques. When a segment is inserted into RF-DNA, half of theresulting phage population will have DNA which includes an insert with asequence which is complementary to that of the insert in the other half.If target DNA or RNA is double-stranded, both types of phage DNA will benucleic acid with sequence of probe. If target DNA or RNA issingle-stranded, only half of the phage DNA will be nucleic acid withprobe sequence. Of course, as the skilled will recognize, the DNAincluding the target segment can be inserted asymmetrically into RF-DNAso that all of the resulting phage population will have DNA with thesame insert and all of the resulting phage DNA will be nucleic acid withsequence of probe. The phage DNA will be treated chemically as describedbelow to transaminate cytosines and link tags to N⁴ -amino groups of thetransaminated cytosines to make a probe of the invention.

RNAs with sequences of probes can be prepared by in vitro chemicaltechniques, by employing suitably protected ribonucleosides in place of2'-deoxyribonucleosides. RNAs with sequences of probes can also beprepared enzymatically by known techniques using suitable DNA templates.

Cytosines in RNAs can be transaminated and subsequently linked to tagusing essentially the same chemistry as described below for DNAs.

Alternatively, rather than first preparing a nucleic acid with asequence of the probe and then transaminating cytosines in the nucleicacid, the nucleic acid with the same sequence as the probe andcomprising an N⁴ -aminocytosine can be made directly by in vitrosynthesis, including enzymatic and solid-phase chemical, using suitableanalogs of N⁴ -aminocytidine or N⁴ -amino-2'-deoxycytidine.

In still a further aspect, the invention provides processes for makingnucleic acid precursors of probes according to the invention bystep-wise, solid-phase syntheses using suitably protected analogs of N⁴-aminocytidine or N⁴ -amino-2'-deoxycytidine. Example III teaches such aprocess utilizing the phosphoramidate chemistry of Matteucci andCaruthers, and Beaucage and Caruthers. In view of the teaching ofExample III, similar processes involving other methods for step-wisechemical synthesis of polynucleotides, in solution or solid-phase, suchas triester methods, will be apparent to the skilled.

Finally, the present invention entails novel, protected analogs of N⁴-aminocytidine and N⁴ -amino-2'-deoxycytidine which are employed in theprocesses provided by the invention for in vitro, step-wise synthesis ofnucleic acid precursors of probes according to the invention. Certain ofthese analogs are described in Example III; from those described, otherswill be apparent to the skilled in the art.

As indicated above, the preferred tag moieties of probes of theinvention are biotin, iminobiotin, sulfenylbiotin, aminothiadiazole,sulfanilamide, PABSAT, fluorescein and tetramethylrhodamine. Mostpreferred are biotin and PABSAT. They are preferably linked to the N⁴-amino group of N⁴ -aminocytosines through a hydrazone linkage and ann-alkyl chain of 2 to 20 carbon atoms, preferably 5 to 8 carbon atoms.

Probes with the hydrazine linkage can be prepared from probes with thehydrazone linkage by simply reducing the probe (with linked tag) afterformation of probe with the hydrazone linkage.

Probes wherein the tag is linked to the N⁴ -amino group of N⁴-amino-cytosines through a linkage of Formula --(C═R₃)NH--, wherein R₃is oxygen or sulfur (preferably sulfur), and a linker moiety --R₅ --(preferably an n-alkylene chain of 2 to 20, more preferably 5 to 8,carbon atoms) are prepared by first making an isocyanate orisothiocyanate-derivatized tag moiety of Formula R₃ ═C═N--R₅ --R₆(wherein R₆ is the tag moiety, preferably one of the preferred eight ofthe invention) and then reacting a quantity of nucleic acid with thesame sequence as the probe and comprising an N⁴ -aminocytosine moietywith the compound of Formula R₃ ═C═N--R₅ --R₆.

Compound of Formula R₃ ═C═N--R₅ --R₆ are prepared by methods known inorganic chemistry by first preparing the amino-derivatized compound ofFormula H₂ N--R₅ --R₆ and then reacting the compound of Formula H₂ N--R₅--R₆ with thiophosgene or phosgene.

Probes with biotin, iminobiotin or sulfenylbiotin tag are detected bycomplexing probe (preferably after hybridization to target) with areporter group, which includes a streptavidin or avidin molecule asconjugate protein to the tag, and then generating a signal from thereporter group. The method of generating a signal depends on whichcompounds besides streptavidin or avidin are included in the reportergroup. Several reporter groups with avidin or streptavidin, for bindingto biotin, are known in the art (see Ward et al., supra; Englehardt etal., supra; Klausner and Wilson, et al., supra) and commerciallyavailable, as from Enzo Biochemicals, Inc., New York, N.Y., U.S.A.,Bethesda Research Laboratories, Inc., Gaithersburg, Md,, U.S.A; andVector Laboratories, Inc., Burlingame, Calif., U.S.A. These groupsinclude acid or alkaline phosphatase complexed with streptavidin oravidin-DH (detection colorimetrically of a product producedenzymatically by the phosphatase), horse radish peroxidase complexedwith streptavidin or avidin-DH (detection colorimetrically of a productproduced enzymatically by the peroxidase), and streptavidin alone(detection fluorometrically of fluorescein-labeled anti-streptavidinantibody bound to the streptavidin). The preferred system is theavidin-DH-biotinylated alkaline phosphatase polymer system described byLeary et al., Proc. Natl. Acad. Sci. (U.S.A.) 80, 4045-4049( 1983).

Avidin-DH is a highly pure grade of avidin available from VectorLaboratories, Inc., Burlingame, Calif., U.S.A. Highly pure avidin fromsources other than Vector Laboratories can also be employed in thepresent invention.

Probes with aminothiadiazole, sulfanilamide or PABSAT as tag aredetected with a reporter group which includes carbonic anhydrase asconjugate protein. Preferred reporter group is polymerized carbonicanhydrase B from a mammalian (preferably bovine) erythrocyte, saidpolymer prepared as described by Epton, Biochem. Soc. Trans. 5,277-279(1977). The preferred method of detecting bound carbonicanhydrase polymer is by the fluorescein diacetate assay of Leary, Anim.Blood Grps. Biochem. Genet. 9, 65-67(1978). See also Livesey, Anal.Biochem. 77, 552-561(1977). Other detection systems (e.g.,immunological, based on antibody-binding to carbonic anhydrase) may beemployed. Further, heteropolymers of carbonic anhydrase with otherenzymes can be employed as reporter group for probes of the inventiontagged with aminothiadiazole, sulfanilamide, or PABSAT. Such otherenzymes include acid and alkaline phosphatase, beta-galactosidase, andhorse radish peroxidase, for which detection systems are well known.See, e.g., Voller et al. "Enzyme-linked Immunosorbent Assay," in Manualof Clinical Immunology, N. Rose and H. Friedman, eds., American Societyfor Microbiology, Washington, D.C., 2nd Fd.(1980). Biotin-labeledcarbonic anhydrase can be employed as reporter group, and all of thesystems described above for detection of biotin through binding ofavidin or streptavidin can be employed to detect the bound carbonicanhydrase.

Probes with fluorescent tag moieties are readily detected directly,without binding of any reporter group, by fluorescence spectroscopyusing methods known in the art. See, e.g., Bauman et al., supra; Baumanet al., Exp. Cell Res. 128, 485-490(1980).

A probe of the invention is used in a hybridization assay for its targetDNA or RNA (and, thereby, the biological entity associated with saidtarget) using standard, known nucleic acid probe hybridization assayprocedures. See Meinkoth and Wahl, supra, and references cited therein.

The assay of a sample will generally be carried out in parallel with anassay of a blank which contains approximately the same amount of nucleicacid as the sample but is known to contain no DNA or RNA with targetsegment for the probe (negative control) and may also be carried out inparallel with an assay of a sample known to contain target DNA or RNA(positive control).

The assay of test sample (or blank or positive control) typicallyproceeds as follows:

Typically, the assay will be carried out on a solid support, such asnitrocellulose paper, to which single-stranded nucleic acid bindsnon-covalently.

Nucleic acid of sample is isolated and affixed to the solid support insingle-stranded form. These isolation and fixation procedures arecarried out so that substantially all of the target segmentcorresponding to probe in any target nucleic acid present in the sampleremains intact.

The solid support then may be prehybridized to substantially eliminatesites available on the support for the non-specific binding of probe.

Then a solution containing probe, in a 10-fold to 10¹² -fold, typicallyabout 10³ to 10⁶ -fold molar excess relative to target segment, isincubated with the solid support under conditions of stringency and fora time sufficient for stable duplex to form between probe and asubstantial fraction (preferably nearly all) of any of its targetsegment on the filter but not to any significant extent between probeand segments other than target segments.

Then unduplexed or partly duplexed probe is washed from the system by aseries of washes (usually 2 or 3) under stringency conditions and overusually short periods of time (minutes) to ensure that substantiallyonly probe stably duplexed to target segment remains in the system.

Those of skill know how to ascertain readily, for a particular probe andsolid support, suitable stringency conditions and time periods for thehybridization and post-hybridization washes that will provide acceptablespecificity and sensitivity for the assays in which the probe will beemployed. See Meinkoth and Wahl, supra.

After unduplexed probe is washed from the system, if reporter group mustbe bound to tag moiety to render probe detectable, a solution ofreporter group conjugate to the tag moiety on the probe is incubatedwith the solid support. Generally, a 10-fold to 10⁶ -fold, usually 10² -to 10⁴ -fold, molar excess of reporter group relative to tag moiety,will be present in the solution and the incubation will be carried outover a time sufficient to ensure that substantially all tag in thesystem is bound by reporter group. Then, unbound reporter group iswashed from the system. Again, the skilled know how to determineconditions of incubation and post-incubation washing to ensure that mosttag in a system is bound by reporter group and that little or noreporter group not associated with tag remains in a system.

The system is then treated appropriately to generate a signal from tagmoiety (if tag moiety not bound by a reporter group renders probedetectable) or reporter group in the system. A determination is thenmade whether such a signal was generated.

If positive control is employed, signal from it is compared with thatfrom blank to ensure that the assay system was functional. If the systemwas functional, the signal from positive control will be greater thanthat from blank.

The signal from test sample is compared with that from blank. If signalfrom test sample is greater than that from blank, target segment waspresent in the sample.

The invention is now illustrated in the following examples:

EXAMPLE I Polynucleotides

The following polydeoxyribonucleotides were synthesized using automatedsolid-phase phosphoramidate methodology (Matteucci and Caruthers, J. Am.Chem. Soc. 103, 3185(1981) and Beaucage and Caruthers, Tetrahedron Lett.1981, 1859-1862) on a Model 380A Applied Biosystems DNA Synthesizer(Applied Biosystems, Inc., Foster City, Calif., U.S.A.). Theoligonucleotides produced with the synthesizer have a free hydroxylgroup at both the 5'-end and the 3'-end. From the mixture ofpolydeoxyribonucleotides of varying lengths produced in a synthesis onthe machine, the desired one, which is the longest, is isolated andpurified by high performance liquid chromatography (HPLC), utilizing alinear gradient of acetonitrile in 0.1M triethyl ammonium acetate (pH7.0) to elute the polydeoxyribonucleotides, as described by Frank etal., Nucl. Acids Res. 11, 4365-4377(1983) at pp. 4369-4370.

Polynucleotide A: 5'-GAAGGGGTATCTTTGGATAAAAG-3'

Polynucelotide B: 5'-CCACCACCTAGAACTAGGATATC-3'

Polynucleotide C: 5 -CCAGGCCAGCCGGAGGGACCCCGGGAGCCCGGGCG-3'

The sequence of Polynucleotide A is complementary to that of a segmentof the alpha-mating factor gene of Saccharomyces cerevisiae. Thesequence of Polynucleotide B is complementary to that of a segment ofthe alcohol oxidase gene of Pichia pastoris. The sequence ofPolynucleotide C is complementary to that of a segment of the genome ofEpstein-Barr virus.

EXAMPLE II Preparation and Characterization of N⁴-Amino-2'-Deoxycytidine

Deoxycytidine was converted to N⁴ -aminodeoxycytidine following theprocedure of Negishi et al., (I) and (II) supra. To 1.0 g (4.4 mmoles)of deoxycytidine (Calbiochem-Behring, La Jolla, Calif. U.S.A.) was added10 ml of 4M hydrazine, 0.1M bisulfite, 0.1M sodium phosphate buffer (toadjust pH to 7.0) and the solution was stirred at 60° C. for 4 hours.Then 90 ml of 95% ethanol was added, and the mixture was allowed to sitat -20° C. overnight. The buffer salts were removed by filtration andthe filtrate was reduced to a thick oil under reduced pressure. Absoluteethanol was added to afford 270 mg (25%) of a semi-solid N⁴-amino-2'-deoxycytidine.

To 10 mg (0.041 mmoles) of N⁴ -amino-deoxycytidine was added 10 mg(0.051 mmoles) of 2,4-dinitrobenzaldehyde in 5 ml of 50% aqueousmethanol. After stirring for 15 minutes at 25° C., the orangeprecipitate which formed was filtered off. The hydrazone was purified bychromatography on silica gel using 10% methanol in chloroform as aneluant. After concentration of the peak fractions under reducedpressure, 12 mg (70%) of N⁴-amino-deoxycytidine-2,4-dinitrophenylhydrazone was isolated andcharacterized by NMR spectroscopy. The product was further characterizedby UV/VIS spectroscopy yielding an extinction coefficient of 18,000 at375 nm (dimethylsulfoxide (DMSO): water, 1:20).

EXAMPLE III Polynucleotides with N⁴ -Aminocytosines

Cytosines in polynucleotides are modified by incubation at 37° C. in amixture of 4M hydrazine with 1M sodium bisulfite buffered to pH 7 with0.1M sodium phosphate, with polynucleotide dissolved to a concentrationsuch that the concentration of cytosines is between about 1.0 and 100micromolar. Under these conditions, the cytosines in a single-strandedpolynucleotide are converted to N⁴ -aminocytosines with pseudofirst-order kinetics with a t_(1/2) of about 30 minutes. Completeconversion occurs in about 4 hours.

In a typical reaction, 200 micrograms of polynucleotide A were incubatedin 0.5 ml of 4M hydrazine, 1M sodium bisulfite, at pH 7.0 (sodiumphosphate buffer) at 37° C. The kinetics of transamination were measuredby two methods. In one, the reaction was stopped after various reactiontimes by separation of hydrazine and bisulfite from the polynucleotideby gel permeation chromatography and then the amount of N⁴-aminocytosine was determined by reaction of 1 volume of apolynucleotide solution with 0.1 volumes of a solution of2,4-dinitrobenzaldehyde (20 mg/ml) in dimethylformamide (DMF) for 30minutes at 23° C., followed by a second gel permeation chromatographystep. The amount of 2,4-dinitrophenylhydrazone-derivatizedpolynucleotide formed was measured spectrophotometrically. In the othermethod, the kinetics were determined by complete digestion of thepolynucleotide with snake venom phosphodiesterase and subsequentanalysis of the monomers by HPLC. Both methods of analysis yielded thesame results. Modification of the polynucleotide was complete within 4hours, and half complete at approximately 30 minutes.

EXAMPLE IV Synthesis of Polynucleotides Using Bases with ModifiedCytosines Directly

Polynucleotide probes can be synthesized by the solid-phasephosphoramidate method of Example I so as to have 4-aminocytosines atspecific locations. In contrast, the procedure of Example III results ina random distribution of modified cytosines in the polynucleotide,governed by the mathematics of the Poisson distribution andnearest-neighbor effects. To synthesize a polynucleotide with N⁴-aminocytosine in place of cytosine at specific locations, suitablyprotected N⁴ -aminocytidine or N⁴ amino-2'-deoxycytidine is used inplace of cytidine or 2'-deoxycytidine at the appropriate steps in theautomated synthesis.

Cytidine or 2'-deoxycytidine is transformed into the corresponding N⁴-amino analog by incubation in a solution of 4M hydrazine and 0.1Msodium bisulfite for 4 hours at 60° C., following the procedure ofExample II (see also Negishi et al. (II), supra). The N⁴ -amino analogis isolated and purified as described by Negishi et al. (II), supra.

The N⁴ -amino-modified nucleosides are then protected for use inautomated synthesis in the same manner as other nucleosides. Inparticular, the N⁴ -amino group, and the 3' and 5' hydroxyls, areperbenzoylated with benzoyl chloride. The 3' and 5' hydroxyls are thenliberated by selective hydrolysis. The 5'-hydroxyl is then tritylatedwith 4'-dimethoxy tritylchloride, and the 3'-hydroxyl isphosphoramidited with methoxymonochloro-N, N-diisopropylaminophosphate.The resulting N-acyl, 5'-trityl, 3'-phosphoramidite derivative ofcytidine or 2'-deoxycytidine is then used in the automated synthesizerin the appropriate steps to synthesize the nucleic acid of desiredsequence. Removal of the benzoyl group from the N⁴ -amino groups of theresulting modified polynucleotide proceeds along with deprotection ofthe other groups upon removal of the polynucleotides from the resin.

EXAMPLE V Biotin-Linker Aldehydes

In this Example, several methods are provided for synthesizingbiotin-linker compounds that can be reacted with polynucleotides,modified to have N⁴ -aminocytosines in place of one or more cytosinemoieties, to attach biotin as a tag moiety through the N⁴ -amino groupof the modified cytosines and thereby provide a probe of the invention.

In the various methods, iminobiotin analogs can be employed in place ofbiotin analogs. Further, any of the biotin linker aldehyde compoundsmade as provided in this Example can be oxidized to sulfenylbiotin byoxidation under mild oxidizing conditions. Both iminobiotin (biotinwherein the oxygen bonded to the ring is replaced with NH) andsulfenylbiotin (biotin wherein the sulfur atom is replaced with asulfenyl group) bind with high affinity to avidin and streptavidin andthereby provide useful tag moieties for the probes of the invention.

(A) Synthesis of Biotin-Linker Aldehydes through an Alcohol Intermediate

This process is illustrated in SCHEME I. ##STR13##

Compound V (6-tertbutyldimethylsilyloxy-1-hexyltrifluoracetamide) wassynthesized as follows:

To 1.00 g (4.8 mmoles) 6-trifluoracetamido-1-hexanol and 0.41 g (6.0mmoles) imidazole in 12.5 ml of dry dimethylformamide (DMF) was added0.91 g (6.0 mmole) tertbutyldimethylsilylchloride at 23° C. The solutionwas allowed to stir for three hours. The solvent was removed and thesample dried under high vacuum. The solid was then extracted with 20%(v/v) ethylacetate in hexane. The organic layer was dried over anhydrousmagnesium sulfate and concentrated to a colorless oil, yielding 1.40 g(89.6%) of Compound V.

The compound of Formula IV (6-tertbutyldimethylsilyloxy-1-hexylamine)was synthesized as follows:

To 1.10 g (3.4 mmole) of compound of Formula V was added 18 mls of asolution of 75% (v/v) methanol in water followed by 0.70 g (5 mmoles) ofpotassium carbonate. The solution was allowed to stir for 15 hours at23° C. The solvent was removed and the solid was extracted with amixture of 20% (v/v) ethylacetate in hexane. The organic layer was driedover anhydrous magnesium sulfate and concentrated to yield 0.72 g (91%)of a pale, yellow oil (Compound IV).

The compound of Formula VI (N-hydroxysuccinimidobiotin) was synthesizedas follows according to the method of Becker et al. (Proc. Natl. Acad.Sci. U.S.A. 68, 2604(1971)):

Biotin (compound of Formula VII), in the D(+) configuration, was used asobtained from Sigma Chemical Company, Inc., St. Louis, Mo., U.S.A.(catalog no. B4501). 240 mg (1.0 mmole) biotin was dissolved in 5 ml ofdry DMF. 210 mg (1.0 mmole) of dicyclohexylcarbodiimide (used aspurchased from Aldrich Chemical Co., Milwaukee, Wis., U.S.A.) and 120 mg(1.2 mmole) of N-hydroxysuccinimide were added to the biotin solutionand the resulting solution was stirred at 23° C. for 15 hours. Theprecipitate that formed was separated by filtration. The filtrate wasthen evaporated under reduced pressure and the resulting residue waswashed twice with ethanol and finally recrystallized from hotacetonitrile to yield 230 mg (64%) of a white crystalline product(compound VI) having a melting point of 216°-218° C.

The compound of Formula III was synthesized as follows:

To 100 mg (0.3 mmole) of N-hydroxysuccinimidobiotin (Compound of FormulaVI) was added 37 mg of N, N-dimethylaminopyridine and 104 mg of6-tertbutyldimethylsilyloxy-1-hexylamine (Compound of Formula IV) in 10ml of dry DMF. The solution was stirred for 16 hours at 23° C. Thesolution was then concentrated and the product isolated by flashchromatography using 20% (v/v) methanol in chloroform. 126 mg (94%) of1-tertbutyldimethylsilyloxy-6-biotinyl-hexylamide (Compound of FormulaIII) was isolated as a white solid. The infrared spectrum of thecompound of Formula III (in KBr) includes peaks at 1642 and 1704 cm¹.The proton nmr spectrum of the compound of Formula III in DMSO-d₆ haspeaks at (shifts in ppm) 0.02 (singlet, 6H), 0.86 (singlet, 9H), 2.99(multiplet, 1H), 3.55 (triplet, 2H), 4.12 (multiplet, 1H), 4.29(multiplet, 1H), 6.35 (singlet, 1H) and 6.41 (singlet, 1H).

All references in the present specification to "flash chromatography"are to the method described by Still et al., J. Org. Chem. 43,2923-2925(1978).

The Compound of Formula II (6-biotinylamide-hexan-1-ol) was prepared asfollows:

Following the procedure of Nakai et al. (Chem. Lett. 1979, 1499), toremove the silyl protecting group, 126 mg (0.29 mmole) of the silylether (Compound of Formula III) was treated with a mixture of aceticacid/water/tetrahydrofuran (3:1:1, v/v) for 24 hours at 23° C. to yieldthe alcohol. 57 mg (60%) of Compound of Formula II was thereby isolated.

An alternative procedure used to prepare the compound of Formula II isas follows:

To 0.5g (1.5mmoles) of Compound VI in 5 ml of DMF was added 0.15g(1.3mmoles) of 6-amino-1-hexanol and the reaction mixture was stirredfor 3 hours at 23° C. After concentration under reduced pressure, andflash chromatography using 20% (v/v) methanol in chloroform, II wasobtained in 94% yield, identical in NMR and IR to the product of theacid hydrolysis of III.

Finally, the biotin-linker aldehyde of Formula CV(6-biotinylamide-hexan-1-al) was prepared as follows:

To 10 mg (30 micromole) of the alcohol of Formula II was added 28 mg (75micromole) of pyridinium dichromate and 50 mg of 3 Angstrom molecularsieves in 10 ml of methylene chloride. After stirring for 3 hours at 23°C., 30 ml of diethyl ether was added to the reaction mixture, which wasthen filtered through a plug of silica gel 60 (Merck grade, obtainedfrom Aldrich Chemical Co., Milwaukee, Wis., U.S.A.). The eluant wasdried and purified by flash chromatography using 10% (v/v) methanol inchloroform. 3 mg (30%) of biotin linker aldehyde of Formula CV wasobtained.

(B) Synthesis through Acid and Acyl Chloride Intermediates

The method of synthesizing biotin-linker aldehyde through acid and acylchloride intermediates is illustrated in Scheme II. ##STR14##

To 250 mg (0.73 mmoles) of N-hydroxysuccinimide biotin (Formula VI) wasadded 0.116 g (0.73 mmoles) of 8-amino octanoic acid in 5 ml of DMF and0.8 ml of 0.1M sodium bicarbonate buffer, pH 8.5. The mixture was warmedto 55° C.-60° C. and allowed to cool to 23° C. After stirring at 23° C.for 14 hours, the solvent was removed under reduced pressure and thesolid crystallized from acetonitrile to give 0.264 g (93%) of8-biotinylamide-1-octanoic acid (Formula X).

8-biotinylamide-1-octanoic acid was converted to the acid chloride(Formula IX) by treatment with oxalyl chloride. To 100 mg (0.26 mmoles)of Compound X was added 0.125 ml (1.44 mmoles) of oxalyl chloride in 6ml of benzene containing 2 drops of DMF. After 30 minutes at 23° C., thereaction mixture was dried in vacuo. The acid chloride was directlyconverted to the aldehyde by adding 20 ml of freshly distilledtetrahydrofuran and 175 mg of 5% palladium on barium sulfate; hydrogengas was bubbled through the mixture for 12 hours. The analysis of thereaction mixture by TLC (thin-layer chromatography) showed severalspots, one of which (Compound of Formula VII) sprayed positive foraldehydes with 2,4-dinitrophenyl hydrazine.

(C) Synthesis of Linker-Aldehyde from Biotin Hydrazide

Another method to make a biotin-linker aldehyde compound is illustratedin Scheme III. ##STR15##

1,10-decane dialdehyde was prepared from the 1,10-decane diol of FormulaXIV as follows:

436 mg (2.5 mmoles) of the 1,10-decanediol was added to 2.26 g (6mmoles) pyridinium dichromate and 2.5 g of 3 Angstrom molecular sievesin 25 ml methylene chloride. After 2 hours of stirring at 23° C., TLCanalysis of the mixture showed complete conversion of the diol to thedialdehyde. The reaction mixture was poured into 100 ml of diethyl etherand this in turn was passed through a plug of silica gel 60. The plugwas then further washed with 50 ml methylene chloride and the organiclayers were combined and then concentrated under reduced pressure to awhite solid. The dialdehyde was then purified by chromatography onsilica gel 60 using chloroform as an eluant. 250 mg (58%) of dialdehydewas isolated as a clear oil.

Biotin-linker aldehyde of Formula CVIII was then prepared frombiotin-hydrazide (Formula XIII) and the 1,10-decane dialdehyde ofFormula XII as follows:

To 70 mg (0.27 mmoles) of biotin-hydrazide (Calbiochem-Behring, Inc.,San Diego, Calif., U.S.A.) was added 175 mg (1 mmole) of the 1,10-decane dialdehyde in 5 ml of 20 parts DMF in 1 part H₂ O (by volume).After stirring for 1 hour at 23° C., analysis of the reaction mixture bythin layer chromatography showed the appearance of product (Compound ofFormula CVIII). Silica gel chromatography using 10% (v/v) methanol inchloroform allowed clean separation of the dialdehyde and the biotinlinker aldehyde. The peak fraction from the chromatograph was pooled anddried to yield 80 mg (72%) of the solid biotin aldehyde (Compound ofFormula CVIII). The infrared spectrum of the compound of Formula CVIII(in KBr) included peaks at 1664 and 1703 cm¹.

When glutaraldehyde was used in place of 1,10-decane dialdehyde in theforegoing procedure, similar results were obtained, with the productcompound having 3 rather than 8 carbon atoms in the alkyl chain linkedto the aldehyde group.

(D) Synthesis of Linker-Aldehyde through Biotin-Linker Acetals

Scheme IV illustrates the synthesis of biotin-linker aldehyde throughbiotin-linker acetal.

The Compound of Formula XVI (6-ethylenedioxy-1-hexylamine) was preparedin three steps from 6-amino-1-hexanol as follows:

To 4 gm (34 mmole) of 6-amino-1-hexanol in 20 ml of 0.05M sodiumbicarbonate, pH 10, was added 5.8 ml (45 mmole) ofS-ethyltrifluorothioacetate with stirring at 23° C. The pH wasmaintained between 9.5 and 10 with 1M NaOH; and, after three hours, theaqueous solution was extracted five times, each with 50 ml ofchloroform. The chloroform solution was dried over anhydrous magnesiumsulfate and concentrated under reduced pressure. Recrystallization fromchloroform afforded 5.6 gms (79%) of product(6-trifluroacetamido-1-hexanol).

To 2.1 gm (10.1 mmole) of the 6-trifluroacetamido-1-hexanol in 40 ml ofmethylene chloride was added 3 gm (10.64 mmole) of pyridinium dichromateand 4.4 gm of 3 Angstrom molecular sieves, and the reaction was allowedto proceed with stirring for 3 hours at 23° C. After addition of 250 mlof ethyl acetate, the reaction mixture was filtered through a small bedof silica gel 60 and concentrated in vacuo. Flash chromatography using50% (v/v) ethyl acetate in hexane afforded 1.48 gms (70%) of6-trifluroacetamido-1-hexanal. ##STR16##

To 1.4 gms (6.8 mmole) of 6-trifluroacetamido-1-hexanal in 21 ml ofmethylene chloride under nitrogen at -78° C. was added 1.8 ml (7.48mmole) of 1,2-bis(trimethylsilyloxy)ethane and 0.14 ml (0.73 mmole) oftrimethylsilyltrifluoromethane sulfonate, and the solution was stirredfor three hours at -78° C., followed by another 0.5 hours at 23° C. Thereaction mixture was taken up in 100 ml methylene chloride, which wasthen washed with 30 ml of saturated sodium bicarbonate solution. Theaqueous layer was extracted with 50 ml of methylene chloride; and theorganic layers were combined, dried over anhydrous magnesium sulfate,concentrated, and flash chromatographed with 50% (v/v) ethyl acetate inhexane to give 1.3 gms (77%) of the product,6-ethylenedioxy-1-hexyltrifluorocetamide.

To 0.380 gm (1.49 mmole) of 6-ethylenedioxy-1-hexyltrifluoracetamide in7.5 ml of 20% (v/v) water in methanol was added 0.3 gm (2.2 mmole) ofpotassium carbonate, and the reaction mixture was stirred for 14 hoursat 23° C. After concentrating the reaction mixture, it was taken up in15 ml of brine (saturated aqueous NaCl) and extracted three times, eachwith 50 ml of diethyl ether, and also four times, each with 50 ml ofchloroform. The organic layers were dried, combined and concentratedunder reduced pressure to give an oil weighing 0.19 gms (80%) which gavea positive color with ninhydrin on a silica gel plate developed in amixture of butanol/acetic acid/water (4:1:1). This oil, which consistsessentially of the compound of Formula XVI, was used directly forsynthesizing the biotin-linker acetal of Formula XV.

Alternately, the same conversion was carried out in quantitative yieldby treating 6-ethylenedioxy-1-trifluoracetamide with 10% piperidine inwater for 45 minutes at 23° C. Lyophilization of the reaction mixtureresulted in complete recovery of the desired compound.

The compound of Formula XV was prepared from the compound of Formula XVIand N-hydroxysuccinimidobiotin (compound of Formula VI, prepared asdescribed in Example VA) as follows:

To 0.090 gm (0.26 mmole) of N-hydroxysuccinimido-biotin in 4 ml of dryDMF were added 0.032 gm (0.26 mmole) of N, N-dimethylaminopyridine and0.05 gm (0.31 mmole) of the 6-ethylenedioxy-1-hexylamine oil (CompoundXVI) and the mixture was stirred for 14 hours at 23° C. Concentration ofthe solution under reduced pressure, followed by medium pressure liquidchromatography on a Lobar pre-packed Si 60 column (from Merck,Darmstadt, West Germany or EM Industries, Inc., Cherry Hill, N.J.,U.S.A.), provided 0.05 gm (50%) of the desired product of Formula XV(biotinyl (N-6-ethylenedioxyhexyl)amide). The following data have beenobtained for the compound of Formula XV: Molecular weight (highresolution mass spectrum): calcd. 385.2035, found 385.2039. Elementalanalysis: calcd. C(56.08), H(8.10), N(10.90); found C(55.78), H(8.09);N(10.72). Proton nmr in DMSO-d₆ (shifts in ppm): 2.04 (triplet, 2H),3.71-3.89 (multiplet, 4H), 4.13 (multiplet, 1 H), 4.30 (multiplet, 1H),4.30 (multiplet, 1H), 4.74 (triplet, 1H), 6.35 (singlet, 1H), 6.42(singlet, 1H), 7.73 (triplet, 1H).

Conversion of compound XV to the aldehyde (CV) was effected inquantitative yield on treatment with 80% aqueous acetic acid at 37° C.for 3 hours, followed by lyophilization.

Alternatively, other acetal groups, in place of that of the compound ofFormula XVI, can be employed as protecting groups in preparing thealdehyde. Such alternative acetal protecting groups are known in the art(see T. Greene (1981), "Protective Groups in Organic Synthesis", Chapter4; Wiley and Sons, N.Y.). Illustrative is a compound of Formula XVIA:##STR17##

In this Example, preparation of PABSAT is described. The preparation isillustrated in Scheme V.

To prepare a halophenyl or nitrophenyl analog of PABSAT, thecorresponding halophenyl or nitrophenyl analog of p-acetamido-benzenesulfonylchloride (compound of Formula XIX) is used in the preparativeprocedure.

To a solution of 0.2 gm (1.1 mmoles) of aminothiadiazole (XX) in 4 ml ofdry pyridine at 0° C. was added, dropwise, a solution of 0.28 gm (1.12mmoles) of p-acetamido-benzenesulfonyl chloride in 3 ml of dry pyridine.The reaction mixture was subsequently warmed to 23° C., and magneticallystirred for 16 hours. After concentration under reduced pressure, thecrude product was dissolved in methanol, and treated with activatedcharcoal, filtered, concentrated under reduced pressure, and purified byflash chromatography using methylene chloride-methanol (65:25) to give0.31 gm of the benzolamide derivative (Compound XVIII) in 75% yield.

To 0.14 gm (0.37 mmole) of Compound XVIII was added 3.5 ml of 1N HCl,and the reaction mixture was refluxed for three hours. On cooling, thesolution was neutralized with ammonium hydroxide, and concentrated underreduced pressure to give a white solid in quantitative yield, which waspure PABSAT (compound of Formula XVII). ##STR18##

EXAMPLE VII PABSAT - Linker Aldehyde

In this Example, methods are described for preparing linker-aldehydecompounds which are suitable for linking PABSAT (and halophenyl andnitrophenyl analogs thereof) to polynucleotides with N⁴ -aminocytosinemoieties and thereby preparing probes of the invention.

The methods of this Example are illustrated in Schemes VIA and VIB.

The methods can also be employed in preparing linker-aldehydes ofaminothiadiazole or sulfanilamide for linking those compounds to N⁴-amino-cytosine-containing polynucleotides to make probes of theinvention. This is accomplished by replacing PABSAT withaminothiadiazole or sulfanilamide in the reaction with the acetal acetylchloride of Formula XXIII in Scheme VIB and preparing theaminothiadiazole or sulfanilamide linker-aldehyde from the resultingaminothiadiazole or sulfanilamide analog, respectively, of the acetal ofFormula XXII. ##STR19##

To 1.1 gm (5.7 mmole) of bromohexanoic acid in 15 ml of dry benzene wasadded 2 ml (23 mmole) of oxalyl chloride and 2 drops of DMF at 23° C.After evolution of gas from the reaction ceased after 10 minutes, thesolution was concentrated in vacuo, and the crude chloride was taken upin 5 ml of dry CH₂ Cl₂. To this solution was added a solution of 0.55 gm(6 mmole) of 2-methyl-2-aminomethyl-1-propanol in 2 ml of CH₂ Cl₂ undernitrogen at 0° C., and the mixture was stirred for 15 minutes, and thenwarmed to 23° C. After concentration in vacuo, excess thionyl chloridewas then added and after 30 minutes the reaction mixture wasconcentrated under reduced pressure. The crude product was purified byflash chromatography using 50% ethyl acetate in hexane to afford 0.75 gm(53%) of very pure oxazoline of Formula XXVI.

To 0.220 gm (1.2 mmole) of 2-(2-bromoethyl)-1,3-dioxolane (compound ofFormula XXVIII) in 1 ml of tetrahydrofuran at -78° C. under nitrogen,was added 1.4 ml (2.4 mmole) of 1.7M solution of tert-butyl lithium inpentane, and the yellow solution was stirred for 15 minutes. Thissolution was transferred via a double-tipped needle under nitrogen intoa flask containing 0.12 gm of cuprous iodide (0.6 mmole) and thereaction mixture was stirred for 30 minutes at -78° C. To the cuprate,compound of Formula XXVII, was added a solution of 0.05 gm (0.2 mmole)of the bromo-oxazole of Formula XXVI in 1 ml of tetrahydrofuran, and thereaction was allowed to proceed at -50° C. for 5 hours, followed bywarming to 23° C. and stirring for 2 hours. Concentration of thereaction mixture in vacuo, followed by solution in methanol andfiltration through a silica gel-60 plug, afforded the crude product.This was purified by flash chromatography using 20% ethyl acetate inhexane to give 0.03 gm (56%) of pure oxazoline-acetal of Formula XXV asa clear oil.

To 0.040 gm (0.15 mmole) of the acetal-oxazoline for Formula XXV isadded 0.5 ml of methyl iodide and the mixture is stirred for 16 hours at23° C. After concentration under a stream of nitrogen, 1 ml of a 1Nsodium hydroxide solution is added, and the mixture is stirred for 15hours at 23° C. After neutralization with 10% hydrochloric acid,followed by extraction with ether, and concentration under vacuum, thedesired acetal-acid of Formula XXIV is obtained.

To 0.030 gm (0.14 mmole) of the acetal-acid of Formula XXIV in 2 ml ofdry benzene is added 0.05 ml (0.56 mmole) of oxalyl chloride and onedrop of DMF. The solution is stirred for 10 minutes and concentratedunder vacuum. The resulting oil (acylchloride of Formula XXIII) is thentaken up in 1 ml of pyridine, and 0.045 gm (0.14 mmole) of PABSAT(Formula XVII) is introduced as a solution in 1 ml of pyridine. Themixture is stirred for 5 hours, and then concentrated in vacuo. Flashchromatography of the crude using 30% methanol in chloroform affords theacetal-linker-benzolamide derivative of Formula XXII.

The compound of Formula XXII is converted to the desired PABSAT linkeraldehyde of Formula XXI by treatment with 80% aqueous acetic acid for 3hours at 37° C. followed by lyophilization.

EXAMPLE VIII Fluorescein-Linker Aldehyde

A mixture of 10.8 mg (0.28 mmoles) of fluorescein isothiocyanate and 23mg (0.14 mmoles) of 6-ethylenedioxy-1-hexylamine in 1.5 ml of drypyridine was stirred at 23° C. for one hour. After concentrating thesolution under reduced pressure, the crude product was purified by flashchromatography using 10% methanol in methylene chloride to afford 11 mg(72%) of fluorescein-linker acetal. This was quantitatively converted tothe aldehyde by treatment with 80% aqueous acetic acid at 37° C. for 3hours followed by lyophilization.

The procedure of the previous paragraph can be carried out with otherisothiocyanate-derivatized fluorescent moieties, such astetramethylrhodamine isothiocyanate and tetraethylrhodamineisothiocyanate, to prepare other fluorescent tag moiety-linkeraldehydes.

The procedure can be carried out also with the isocyanate derivatives ofthe fluorescent moieties in place of the isothiocyanate derivatives. Theisocyanate derivatives, like the isothiocyanate, are known.

EXAMPLE IX Probes with Biotinyl, Iminobiotinyl or Sulfenylbiotinyl asTag Moiety

To prepare a probe of the invention with biotinyl, iminobiotinyl, orsulfenylbiotinyl as tag moiety, using a linker aldehyde compoundprepared in accordance with Example V, the following procedure isemployed:

100 micrograms of a polynucleotide with the sequence of the probe istreated as described in Example III to transaminate (convert to N⁴-aminocytosine) about 10% to about 50% of the cytosines in thepolynucleotide. The reaction is stopped by passing the solution througha Sephadex G-25 spin column and isolating the modified polynucleotide bycollecting the effluent. To the effluent (containing approximately 75microgram (ug) of polynucleotide in approximately 150 microliters (ul))is added 40 ul of linker compound solution (5 mg/ml in DMSO). After 30minutes at 37° C., the mixture is passed through a Sephadex G-25 spincolumn and tagged polynucleotide is obtained in the collected effluent.

Concentration of the tagged probe is quantitated by UV absorbance at 260nm and biotinylation is confirmed by spotting 1 ul of the probe solutionon nitrocellulose paper and detecting the probe as described by Leary etal. (1983), supra., using avidin-DH and biotinylated alkalinephosphatase polymer.

EXAMPLE X Probes With PABSAT, Aminothiadiazole or Sulfanilamide as TagMoiety

To prepare probe of the invention with PABSAT, aminothiadiazole orsulfanilamide as tag, the following procedure is employed:

First, following the procedure of Example III, a polynucleotide with thesequence of the probe is modified to convert about 10 to about 50% ofits cytosines into N⁴ -aminocytosines. The modified polynucleotide(approximately 75 ug in approximately 150 ul) is then mixed with 50 ul(5 mg/ml in DMSO) of a solution of sulfonamide linker aldehyde (e.g.,compound of Formula XXI) prepared in accordance with Example VII. After30 minutes at 37° C., unreacted aldehyde is separated from labeled probeby spin column chromatography, as in Example IX.

Concentration of tagged probe is determined by UV absorbance at 260 nm.Attachment of tag is confirmed by detecting carbonic anhydrase polymerreporter group bound to tag, following procedures in Example XIII.

EXAMPLE XI Probes with Fluorescein or Tetramethylrhodamine As Tag Moiety

To prepare probe of the invention with fluorescein ortetramethylrhodamine as tag moiety, the following procedure is employed:

First, following the procedure of Example III, a polynucleotide with thesequence of the probe is modified to convert between about 10% to about50% of its cytosines into N⁴ -aminocytosines. The modifiedpolynucleotide (approximately 75 ug in approximately 150 ul) is thenmixed with 50 ul of a solution (5 mg/ml in DMSO) of a fluorescein-linkeraldehyde or tetramethylrhodamine-linker aldehyde prepared in accordancewith Example VIII. After 30 minutes at 37° C., unreacted aldehyde isseparated from labeled probe by spin column chromatography, as inExample IX.

Concentration of tagged probe is determined spectroscopically.Attachment of fluorescent tag is confirmed by fluorescence emissionspectroscopy.

EXAMPLE XII Use of Probes of the Invention in Nucleic Acid HybridizationAssays

The competency of probes of the invention in hybridization assays wasestablished in a Southern hybridization protocol.

Polynucleotide B (Example I) was transaminated according to Example IIIin a reaction lasting 10 minutes, so that, on the average, between 1 and2 cytosines per molecule of polynucleotide were converted to N⁴-aminocytosine. The N⁴ -aminocytosine-containing polynucleotide was thenbiotinylated according to Example IX with the linker aldehyde of FormulaCV. The biotinylated polynucleotide B was used in the Southern protocol.

A 730 base pair HindIII-SalI DNA fragment, which is a portion of thecoding segment of the alcohol oxidase gene, was isolated from Pichiapastoris by standard procedure. See Ellis et al., Mol. Cell. Biol. 5,1111-1121(1985). By standard procedures, this fragment was cloned inboth M13mp18 and M13mp19 in E. Coli JM103. The M13mp18 clones producephage with DNA with a segment identical in sequence to Polynucleotide B.The M13mp19 clones produce phage with DNA with a segment with a sequencecomplementary to that of Polynucleotide B.

Large quantities of each type of phage were prepared by standard methodswith E. coli JM103. After purification, DNAs from the M13mp18 andM13mp19 phage were attached to separate nitrocellulose filters by meansof Manifold II Slot Blotter apparatus (Schleicher and Schuell, Inc.,Keene, N.H., U.S.A.). As a positive control, a denatured,double-stranded E. coli plasmid, pUC19, was also attached to a filter bythe same method. On each filter, the DNA was aliquoted in bands of 500ng, 100 ng, 10 ng, 1 ng, 100 pg, 10 pg, and 1 pg.

Attachment was followed by baking in a vacuum oven at 80° C. The filterswere then prehybridized for 2 hours at 42° C. with 6×SSPE, 0.5% (w/v)sodium dodecyl sulfate (SDS), 1× Denhardt's solution, 1 mg/ml herringsperm DNA. Following the prehybridization, the filters were hybridizedwith probe as follows:

    ______________________________________                                        Filter 1:       probed with 1 ug                                              (M13mp19 DNA)   of biotinylated                                                               Polynucleotide B, at                                                          300 ng/ml in hybridization                                                    solution.                                                     Filter 2:       probed with                                                   (M13mp18 DNA)   1 ug of biotinylated                                                          Polynucleotide B, at                                                          300 ng/ml in hybridization                                                    solution.                                                     Filter 3:       probed with 300 ng pUC19 DNA                                  (pUC19 DNA)     nick-translated with a                                                        biotinylated                                                                  2'-deoxyuridine-5'-                                                           triphosphate, essential                                                       according to the procedure of                                                 Langer et al., Proc. Natl.                                                    Acad. Sci.(U.S.A.) 78,                                                        6633-6637(1981) (using a                                                      nick-translation kit                                                          purchased from Bethesda                                                       Research Laboratories,                                                        Gaithersburg, Maryland,                                                       U.S.A.) and at 100 ng/ml in                                                   hybridization solution.                                       ______________________________________                                    

Hybridizations were done at 42° C. for 15 hours. Hybridization solutionwas 6×SSPE, 0.5% (w/v) SDS, and 1× Denhardt's solution. After thehybridization, the filters were washed as follows:

    ______________________________________                                        Filter 1:    2 × SSC for 15 minutes, twice, at                                       room temperature                                                              2 × SSC for 15 minutes, once, at                                        room temperature                                                 Filter 2:    Same as filter 1                                                 Filter 3:    2 × SSC and 0.1% (w/v) SDS for                                          15 minutes, twice, at room                                                    temperature                                                                   2 × SSC and 0.1% (w/v) SDS for                                          15 minutes, twice, at 42° C.                                           2 × SSC for 15 minutes, once, at                                        room temperature                                                 ______________________________________                                    

Definitions of SSC, SSPE, and Denhardt's Solution are known in thenucleic acid hybridization art. See Maniatis et al., Molecular Cloning:A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y., U.S.A.(1982).

The filters were then developed with streptavidin-biotinylated alkalinephosphatase polymer as reporter group, prepared according to theprocedure of Leary et al. (1983), supra, using substrate of alkalinephosphatase with associated dye for colorimetric indication, alsoaccording to the procedure of Leary et al.(1983), supra.

Color development was monitored after 1.5 hours.

The test filter (Filter 1) showed hybridization down to the band with100 pg DNA. The negative control filter (Filter 2) showed nohybridization. The positive control filter (Filter 3) showedhybridization down to the band with 1 pg DNA.

EXAMPLE XIII Use of Aminothiadiazole-Benzolamide Derivatized Probes inHybridization Assays

In this Example, use of probes tagged with aminothiadiazole-benzolamidederivatives, such as PABSAT, is described.

Following the procedure of Example IV, Polynucleotide C is synthesizedto have N⁴ -aminocytosines at positions 6, 20, 28, and 34 (from the5'-end).

Following the procedure of Example X, using PABSAT linker aldehyde ofFormula XXII prepared as described in Example VII, the N⁴-aminocytosine-containing Polynucleotide C is converted to probe withPABSAT linked to the N⁴ -amino nitrogens of the modified cytosines.

DNA is isolated from two mammalian cell cultures, one known to beinfected with Epstein-Barr virus (EBV) and the other known to beEBV-free. Approximately 5 ug of protein-free DNA from each culture isaffixed to a separate, prewetted nitrocellulose filter using standardblotting techniques.

The filters are then prehybridized as described in Example XII.

Each filter is then hybridized with 1 ug of PABSAT-derivatizedpolynucleotide C, as described in Example XII, at 300 ng/ml inhybridization solution.

Following the hybridization, the filters are washed, also as in ExampleXII.

The filters are then developed with a reporter system based on thefluorescein-diacetate assay of Leary et al.(1978), supra, for bovineerythrocyte carbonic anhydrase B as follows:

Bovine erythrocyte carbonic anhydrase B is purchased from Sigma ChemicalCo., St. Louis, Mo., U.S.A. and purified as described by Armstrong etal., J. Biol. Chem. 244, 5137-5149(1966). The purified protein ispolymerized by the procedure of Epton, supra.

The filters with hybridized probe are incubated for 5 minutes at roomtemperature with a solution of protein polymer (20 ug/ml) in Tris buffer(pH 7.6). After the incubation, the filters are washed 5 times with0.05M potassium phosphate buffer, pH 6.8, to remove protein polymer thathas not bound to probe through PABSAT tag.

Finally, the filters are incubated for 4 hours at room temperature witha solution of 1 mM fluorescein diacetate in 0.05M potassium phosphatebuffer, pH 6.8.

After the incubation with fluorescein diacetate, the filter with EBV DNAexhibits a fluorescent yellow-green color while the other filterexhibits no fluorescence.

While the foregoing examples illustrate the present invention, they arenot intended to limit the scope thereof. The skilled in the art willrecognize, from the exemplified embodiments, modifications andvariations that are within the spirit and scope of the inventiondescribed and claimed herein.

What is claimed is:
 1. A compound of Formula XXXV: ##STR20## wherein R₆₀is alkylene of 2 or 3 carbon atoms; and wherein k is 2 to
 20. 2. Acompound according to claim 1 wherein k is 5 to
 8. 3. A compoundaccording to claim 2 wherein R₃₂ is ##STR21##